使用溶液式原子力顯微鏡探討大腸桿菌接合生殖中DNA的傳遞

在本研究中，利用溶液式原子力顯微鏡(liquid atomic force microscopy, Liquid AFM)研究大腸桿菌(Escherichia coli, E. coli)授株(donor strain) AT2453與受株(recipient strain) AB1157接合生殖作用(conjugation)中DNA的傳遞機制。與其他高解析度的顯微鏡技術相比，例如掃描電子顯微鏡(scanning electron microscopy, SEM) 以及穿透式電子顯微鏡(transmission electron microscopy, TEM)，AFM最令人驚豔的優勢在於其能讓生物樣品處於生理條件下(physiological condition)進行觀察與研究。AFM系統同時也可以作為極為精準的力量測儀器。因此可利用AFM力譜(AFM force spectroscopy)，藉由經ssDNA抗體(anti-single-stranded DNA antibody)修飾之AFM探針，研究活體大腸桿菌接合生殖中DNA的傳遞機制。在本研究中，將培養至對數生長中期(mid-log growth phase)的大腸桿菌AT2453與AB1157固定在蓋玻片上。先利用AFM探針對介於大腸桿菌AT2453與 AB1157接合生殖對(mating pair)之間的性線毛(sex-pilus, 亦稱F-pilus)進行成像後再加以切割，而後再以anti-ssDNA antibody修飾過之AFM探針，對已被切割的性線毛切口處進行力量測。為了與上述比較，也對完整的性線毛上或遠離切口處的區域進行力量測。另外，更對純ssDNA樣品進行力量測，用以進一步確認單一ssDNA與anti-ssDNA antibody間鍵結力的大小。根據本研究之AFM力譜量測結果，只有在性線毛切口處發現量測到DNA，其量測到的力大小為109 ± 5 pN。也因此本研究提供一個直接的證據，證明大腸桿菌接合生殖時，是經由大腸桿菌接合生殖對間的性線毛中空管狀通道進行DNA的傳遞。

關鍵字

大腸桿菌；接合生殖；原子力顯微鏡

並列摘要

The mechanism of DNA-transfer from Escherichia coli (E. coli) Hfr donor strain AT2453 to recipient strain AB1157 during conjugation process has been investigated by liquid atomic force microscopy (AFM) in this work. Compared with other techniques such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the most fascinating advantage of AFM as a high-resolution microscope is that it allows investigations and observations of biological species in physiological conditions. The AFM system can also be used as an ultra-sensitive force-measurement apparatus as reported earlier. By decorating anti-ssDNA (anti-single-stranded DNA) antibody onto AFM probes, we can utilize the AFM force-spectroscopy and the specific binding between ssDNAs and anti-ssDNA antibody to detect the presence of ssDNA, so as to investigate the DNA transfer mechanism during E. coli conjugation in vivo. In this study, both E. coli AT2453 and AB1157 were cultured and then immobilized on glass coverslip. The F-pilus between the E. coli AT2453 and AB1157 mating pair was imaged by an AFM probe and then dissected. Another AFM probe decorated with an anti-ssDNA antibody was applied to detect the ssDNA in the dissected area and measure the binding force between ssDNA and its antibody. As comparisons, the force-measurements were also conducted on undamaged F pilus or regions away from the dissected area. In addition, the measurements on pure ssDNAs were carried out to further confirm the value of binding force between ssDNA and anti-ssDNA antibody. According to the AFM force spectra, the transferred-ssDNA was only detected in the dissected area with a binding force of 109 ± 5 pN measured. Our results provide direct evidence that the DNA was transferred through the F-pilus channel between an E. coli mating pair during their conjugation.

並列關鍵字

E. coli ； Escherichia coli ； Conjugation ； Atomic Force Microscopy

參考文獻

1. Medigue, C., Rouxel, T., Vigier, P., Henaut, A. and Danchin, A. Evidence for horizontal gene transfer in Escherichia coli speciation. J. Mol. Biol. 222, 851-856 (1991).

2. Ippen-Ihler, K.A. and Minkley, E.G., Jr. The conjugation system of F, the fertility factor of Escherichia coli. Annu. Rev. Genet. 20, 593-624 (1986).

3. Gelvin, S.B. Agrobacterium-Mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol. Mol. Biol. Rev. 67, 16–37 (2003).

4. Harrington, L.C. and Rogerson A.C. The F pilus of Escherichia coli appears to support stable DNA transfer in the absence of wall-to-wall contact between cells. J. Bacteriol. 172, 7263–7264 (1990).

5. Samuels, L., Lanka, E., and Davies, J.E. Conjugative junctions in RP4-mediated mating of Escherichia coli. J. Bacteriol. 182, 2709–2715 (2000).

國際替代計量

使用溶液式原子力顯微鏡探討大腸桿菌接合生殖中DNA的傳遞

全文下載

主題瀏覽